Signal transduction from toll-like receptors (TLRs) mediates innate immune cell activation in host defense against infections; however, deregulated TLR signaling also contributes to the development of inflammatory diseases. The long-range goal of this research project is to understand the molecular mechanisms regulating TLR signaling in macrophages, a major type of innate immune cells involved in the regulation of immune responses, inflammation, as well as tumor microenvironment. During the previous funding cycles, the PI's laboratory has made seminal discoveries in this area. Moreover, we have generated a large body of innovative preliminary data that form a solid foundation for this continuation application. In particular, our preliminary studies identified a novel TLR signaling mediator, Zranb1 (also called Trabid). Zranb1 is a deubiquitinase with poorly defined physiological functions due to the lack of an animal model. Using newly generated Zranb1 conditional KO mice, we found that Zranb1 deficiency in macrophages specifically attenuates TLR-stimulated expression of a subset of proinflammatory cytokines, including IL-12 and IL-23. We have obtained preliminary evidence that Zranb1 regulates the fate of c-Rel, a transcription factor involved in IL-12/IL-23 gene induction. Along the same line, our preliminary studies led to the unexpected observation that a TRAF family member, TRAF2, serves as a pivotal negative regulator of the proinflammatory axis of TLR signaling. TRAF2 aberrant expression is associated with human inflammatory bowel disease (IBD), but it has been unclear whether TRAF2 positively or negatively regulates colon inflammation. By generating myeloid cell-conditional Traf2 KO (Traf2-MKO) mice, we demonstrated that TRAF2 ablation greatly promotes TLR-stimulated proinflammatory cytokine expression in macrophages and sensitizes mice for colitis induction in an animal model of IBD. We further showed that TRAF2 deficiency in macrophages leads to marked accumulation of c-Rel and IRF5, major transcription factors mediating induction of proinflammatory cytokines. Interestingly, our data suggest the regulation of c-Rel and IRF5 by a novel ubiquitin/proteasome-dependent mechanism relying on both TRAF2 and a related TRAF member, TRAF3. Collectively, these findings establish TRAF2 and Zranb1 as pivotal regulators of TLR signaling and highlight a novel signaling mechanism. Our hypothesis is that TRAF2 and Zranb1 regulate the proinfammatory axis of TLR signaling through controlling the fate of important signaling molecules, including c-Rel and IRF5. To test this hypothesis, we will (1) elucidate the mechanism by which TRAF2 regulates TLR signaling in macrophages; (2) define the molecular mechanism by which Zranb1 mediates TLR signaling; and (3) investigate the in vivo pathophysiological functions of TRAF2 and Zranb1 in myeloid cells. We believe that the proposed project addresses a unique and novel aspect of TLR signaling and will yield innovative results that substantially advance the field.